Calcium (Ca2+) is one of the most important intra- and intercellular messengers in eukaryotes, where it regulates many essential processes, including innate immune responses. Therefore, abnormalities in cellular Ca2+ homeostasis have been implicated in many diseases, including pulmonary and heart diseases that are commonly associated with bacterial infections. In bacteria, Ca2+ has been shown to affect various physiological processes including virulence. However, the molecular mechanisms of this regulation are still elusive. Identification and characterization of proteins involved in Ca2+ regulatory and signalling pathways will not only provide a missing direct evidence of the signalling role of Ca2+ in prokaryotes, but more importantly, will provide a new understanding of Ca2+-regulated host-pathogen interactions triggering the development of infectious diseases. In the proposed research we will study Pseudomonas aeruginosa, a facultative human pathogen that is a leading cause of severe chronic infections in cystic fibrosis (CF), endocarditis, wound, burn, and implant patients. Earlier we showed that elevated Ca2+ enhances the production of virulence factors and infectivity in P. aeruginosa. We identified a putative Ca2+-binding protein, EfhP, which is homologous to calmodulin and contains two canonical EF-hand motifs. We hypothesize that EfhP binds Ca2+ and plays a key role in Ca2+ signaling and regulation of virulence. To test the hypotheses we will 1) measure Ca2+ binding by EfhP and identify the required amino acid residues; 2) characterize the role of EfhP Ca2+ binding in regulation of virulence factors; 3) characterize the transcriptional profile of efhP in response to Ca2+, other host-related factors, in clinical isolates and CF sputum samples; 4) identify the regulatory phosphorelay pathways triggered by EfhP Ca2+-binding; and 5) characterize the role of EfhP and Ca2+ in P. aeruginosa-host interactions. The research is innovative as it will be first to study the relationship between Ca2+-binding protein, Ca2+ signaling and Ca2+-regulated virulence in a human pathogen of extremely high importance. Understanding the molecular mechanisms by which Ca2+ regulates P. aeruginosa virulence will advance the current understanding of infectious diseases associated with Ca2+ imbalance and eventually provide novel targets for developing new antimicrobial drugs. The proposed research will be performed by teams of undergraduate and graduate students. It is designed to provide an excellent training environment for students by ensuring their involvement in modern research, their participation in scientific meetings and professional networking, and their publications in peer-reviewed journals.